Inflammatory Status of Excavated Pulp Tissue and Internal Root Resorption in Pulpotomized Primary Molars

Statement of the Problem: Internal root resorption after pulpotomy is a pathological phenomenon and can lead to early root resorption and subsequent loss of the tooth Purpose: To assess the relationship between initial inflammatory coronal pulp status in decayed primary molars treated by pulpotomy and internal root resorption after one-year follow-up. Materials and Method: In this clinical in vivo and in vitro experiment, vital pulpotomies were performed on 50 primary molars from 50 patients aged 5 to 10 years. Coronal pulp was carefully removed followed by hemostasis and placement of a reinforced zinc oxide eugenol over the vital radicular pulp. Enzyme-linked immunosorbent assay (ELISA) assay was done on coronal pulp samples and the level of tumor necrosis factor-alpha (TNF- α) and interlukin-6 (IL-6) was measured. After a 12-month follow-up, periapical radiographs were taken from pulpotomized teeth. Kolmogorov-Smirnov, Chi-square, Kruskal–Wallis, and Mann-Whitney tests were implemented. Results: 11 treated teeth (22%) showed an internal root resorption as diagnosed on X-rays. No significant association was found between TNF- α, IL-6 levels, and pathological root resorption respectively (p= 0.953) and (p= 0.944). A significant association between age and pathological root resorption was observed (p= 0.031). No significant association between remaining dentin thickness and pathological root resorption was established (p= 0.346) Conclusion: There was no association between pro-inflammatory cytokines levels/ TNF-α, IL-6 and internal root resorption following pulpotomy in pediatric patients.


Introduction
Pulpotomy is a common pulp therapy on carious primary molars [1]. The coronal pulp is removed and hemostasis is achieved at the level of pulp stumps. The radicular parenchyma, supposedly healthy, is protected with a capping material [1].
Caries lesions affect the inflammatory status of the pulp and involve the enrolment of immunocompetent and inflammatory blood cells. Pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interlukin-6 (IL-6) are released in response to the compo-nents of caries-related bacteria. Elevated levels of IL-6 were found in carious deciduous molars with exposed pulp [2]. The increase in the level of these polypeptides in pulpal blood may help in diagnosis of pulpal inflammation and makes them possible to be used as markers for assessing pulpal status [2][3][4][5].
The treatments' success or failures on vital teeth with deep carious lesion are assessed based on radiological and clinical signs and symptoms. Internal root resorption (IRR) after pulpotomy is caused by chronic inflammation of the remaining radicular pulp [6]. For Smith et al. [7] this process is not a failure since it is not revealing any osseous change or injuries to the permanent successor. For other investigators, IRR is an obvious sign of failure even without clinical manifestations or osseous alterations [8]. The diagnosis of internal root resorption is mainly based on radiographs with supplementary information gained from history and clinical findings. Contrary to the external root resorption, IRR presents on X-rays a well-defined margin and a symmetrical lesion [9].
The inflammatory role of cytokines in bone resorption and in all disease patterns associated with osteonecrosis is well defined [10]. Many authors elucidate their impacts on physiological root resorption, on pathological external root resorption during orthodontic tooth movement, and on the process of periodontal tissue remodeling [11][12][13][14]. Moreover, cytokines levels are associated with pulp pathogenesis [2].
This study aims to find a correlation between inflammatory cameral pulp status and IRR after 12 months' follow-up pulpotomy treatment on carious primary molars. We hypothesized that pathological root resorption after pulpotomy could be related to elevated levels of cytokines (IL6 and TNF-α) on the coronal pulp. Follow-up examinations of the pulpotomized teeth and healthy ones were carried out after 12 months.

Materials and Method
The collected sample was transferred into a single well of an IL-6/TNF-α assay plate supplied by the manufacturer (R&D System Bio-Techne) and the Enzymelinked immunosorbent assay (ELISA) was applied.
Statistical analysis was conducted using IBM SPSS version 24 (IBM Corp., Armonk, N.Y., USA). Descriptive statistics were presented as mean ± standard deviation for continuous variables and as frequencies and percentages for categorical variables. Kolmogorov-Smirnov, Chi-square, Kruskal-Wallis, and Mann-Whitney tests were done. Level of significance was set below p= 0.05.

Results
A total of 50 primary molars /19 second primary molars and 31 first primary molars, with deep dentinal caries and a value of remaining dentin thickness ranged from 0.3mm to 1mm, requiring vital pulpotomy, were included in this study ( Table 1). The distribution of patient age and gender is outlined in Table 2. From 50 selected patients aged between 5 and 10, mean age=6.8 years (Table 1), 22 were female and 28 males (Chi-square test, p= 0.480), (Table 2) and distributed as follow: 6 patients were 5 years old, 22 between 6 and 8 years and 22 from 8 to 10 years (Chi-square test, p= 0.006), (Table 2).

Discussion
In this research, the inflammatory coronal pulp status was appraised as a factor affecting the internal root resorption. Assessments were made based on the levels of inflammatory results as well as periapical radiographic evaluation.
The proximity of carious lesions to the pulp was measured on preoperative X-rays according to the technique applied by Berbari et al. [15]. To detect and to measure the pro-inflammatory cytokines, the assay by the high sensitivity ELISA technique was considered as a validated method [3]. The number chosen for our samples (n= 50) was enough to carry out the statistical analysis as well to determine the significance of the results [16]. To minimize the patient's exposure, 12 months of follow-up seemed an acceptable period to evaluate the treatment's outcomes [17].
To rule out the factors that could interfere with the results, a well-established methodology was carried out by a single operator. Moreover, copious irrigation was done to avoid tissues' necrosis and to enhance the healing capacity of the residual vital pulp. After pulpal trea- tment, a stainless-steel crown was immediately wellsealed to prevent leakage and thereby bacterial infiltration and inflammation of the remaining pulp and possible occurrence of IRR [18][19][20].
The American Academy of Pediatric Dentistry [20] recommended pulpotomy in primary tooth with extensive caries. For Ghaderi et al. [21] caries extension measured on radiographs could be a potential diagnostic tool to pulpotomy treatment. In this study, the inclusion criteria required a mean value of remaining dentin thickness between 0.3mm and 1.0mm (Table 4). According to Berbari et al. [16], this element may prove itself as an additional indicator for pulpotomy.  (Table 4). Conversely, with a low level of cytokines, some cases presented a pathological root resorption (samples #12, #24, #48), (Table   4). These contrasting results could be justified by many different etiological factors such as an erroneous pulp diagnosis before treatment decision [24]. In fact, establishing a correct diagnosis is essential to obtain high success rates in pulpotomy treatment. Assessing the pulpal status of primary teeth can be difficult since children are not capable to give detailed history of symptoms. Moreover, the exact pulp diagnosis could not be consistent with the severity of the symptoms [25].
During pulpotomy, pulp diagnosis is based on radiological evaluation and clinical signs. The color, the aspect and the intensity of pulp bleeding were considered as predictable factors for pulpotomy success [21,26].
Conversely, recent studies showed no direct correlation between successful hemorrhage control and radicular pulp inflammation [27]. In the study of Fuks et al. [28], the intraoperative diagnosis/ bleeding time and color, although clinically reliable, did not precisely determine the condition of the pulp tissue. In this research, in line with the results of this study, elevated level of cytokines was observed in some pulp samples (#27, #30, #36) even though all the pulpotomies were performed within the hemostasis time ( Table 4).
The formation of a blood clot after pulpotomy, followed by a chronic inflammation of the residual pulp, might be an additional causal factor for internal root resorption [29][30]. The placement of the material over a bleeding pulp or a clinically observable blood clot is favorable for microbial development. According to Shröder [30], extra-pulpal blood clot between the wound surface and the dressing materiel would increase the initial inflammatory response originated by a pulpotomy agent, due to the chemotactic effect of the polymorphonuclear leukocytes. To dissolve any extra-pulpal blood clot, sodium hypochlorite was suggested [31]. In the current study, the hemostasis was achieved by a cotton pellet soaked with sterile saline. Thus, could be one of the main reasons of internal root resorption.
The wound dressing material is an important factor affecting the outcome of the pulpotomy. The ideal capping material should be bactericidal, harmless to cells, and promoting the healing of pulp tissue without interfering with the physiological root resorption [32][33].
According to Ratnakumari and Thomas [32], the pulp response is the best criterion to judge the performance of a capping agent. To evaluate the relationship between cytokines and pulpotomy agents, Lourenço Neto et al. [34] remarked that clinical and radiographic successes are not consistent with the true state of pulp health. So far, the presence of an ideal pulp dressing material is not evident [35]. In this current study, zinc oxide-eugenol with polymer reinforcement was used knowing to have decreased irritating effects of eugenol and the presence of polymethylmethacrylate [36]. This product was applied without any fixative, preservative or astringent agent, while maintaining the integrity of the root pulp.
In the study of Hui-Derksen et al. [37] the overall success rates were obtained after vital pulp pulpotomy with reinforced zinc oxide-eugenol made this latter a reliable procedure for non-chronically inflamed primary molars. Moreover, a low rate of internal resorption (~2%) was observed [37].  [38][39][40]. The results of the study of Sönmez and Durutürk [8] showed that IRR was not affected by physiological root resorption.
The present study showed no significant association between remaining dentin thickness and pathological root resorption (p= 0.346), (Table 3). In fact, a lesion very close to the pulp does not induce necessarily a pathological root resorption (samples #4, #7), (Table 4).
On the other hand, a pathological resorption was ob-served in shallower caries (samples #35, #44), (Table   4). When performing a pulpotomy, regardless of the numerical value of the caries depth, many other factors should be considered to avoid treatment's failure. The stage of caries lesion activity, the thickness and degree of calcification of the remaining dentine, the nature and aspect of the residual dentin, and especially the reactionary dentin or sclerotic dentin should be carefully examined [16,21].
This original article showed for practitioners the relationship between initial inflammatory coronal pulp status and internal root resorption. When performing pulpotomy in carious primary molars, dentists should always keep in mind all factors leading to pathological root resorption even if this latter was not clinically observed. This awareness saves them from being tempted to think of a causal relation. Moreover, this subject opens for researchers the opportunities to deal with such topic again.
The current study was not without limitations; the overlapping of the dental and bone structures in maxilla made radiological observation difficult and some inflammatory cytokines were not studied. An increase in sample size and follow up period would lead to results that are more accurate. Further research is warranted to study the association between immune pulp status and internal root resorption.

Conclusion
In this research, TNF-α and IL-6 levels were evaluated as factors affecting the outcomes of pulpotomy treatment. The study found no association between inflammatory coronal pulp status and internal root resorption following pulpotomy. Internal root resorption diagnosed on radiography after pulpotomy could be unnoticed clinically.

Conflicts of Interest
The authors declare that they have no conflict of interest.